Search tips
Search criteria

Results 1-25 (723145)

Clipboard (0)

Related Articles

1.  Isolation and characterization of a suppressor mutation that restores Myxococcus xanthus exopolysaccharide production 
Microbiology  2009;155(Pt 11):3599-3610.
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
PMCID: PMC2879065  PMID: 19684067
2.  Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus▿  
Journal of Bacteriology  2010;192(17):4267-4274.
Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P.
PMCID: PMC2937368  PMID: 20543066
3.  A CheW Homologue Is Required for Myxococcus xanthus Fruiting Body Development, Social Gliding Motility, and Fibril Biogenesis 
Journal of Bacteriology  2002;184(20):5654-5660.
In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.
PMCID: PMC139594  PMID: 12270823
4.  Myxococcus xanthus Chemotaxis Homologs DifD and DifG Negatively Regulate Fibril Polysaccharide Production 
Journal of Bacteriology  2004;186(4):1001-1008.
The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium. These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility. Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus. It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development. Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG. difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively. difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions. Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development. The difB mutant showed no obvious defects in any of the processes examined. In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type. The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M. xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis. To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria. In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M. xanthus.
PMCID: PMC344214  PMID: 14761994
5.  Nitrate-Dependent Activation of the Dif Signaling Pathway of Myxococcus xanthus Mediated by a NarX-DifA Interspecies Chimera 
Journal of Bacteriology  2005;187(18):6410-6418.
Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.
PMCID: PMC1236652  PMID: 16159775
6.  DifA, a Methyl-Accepting Chemoreceptor Protein-Like Sensory Protein, Uses a Novel Signaling Mechanism to Regulate Exopolysaccharide Production in Myxococcus xanthus▿ †  
Journal of Bacteriology  2010;193(3):759-767.
DifA is a methyl-accepting chemotaxis protein (MCP)-like sensory transducer that regulates exopolysaccharide (EPS) production in Myxococcus xanthus. Here mutational analysis and molecular biology were used to probe the signaling mechanisms of DifA in EPS regulation. We first identified the start codon of DifA experimentally; this identification extended the N terminus of DifA for 45 amino acids (aa) from the previous bioinformatics prediction. This extension helped to address the outstanding question of how DifA receives input signals from type 4 pili without a prominent periplasmic domain. The results suggest that DifA uses its N-terminus extension to sense an upstream signal in EPS regulation. We suggest that the perception of the input signal by DifA is mediated by protein-protein interactions with upstream components. Subsequent signal transmission likely involves transmembrane signaling instead of direct intramolecular interactions between the input and the output modules in the cytoplasm. The basic functional unit of DifA for signal transduction is likely dimeric as mutational alteration of the predicted dimeric interface of DifA significantly affected EPS production. Deletions of 14-aa segments in the C terminus suggest that the newly defined flexible bundle subdomain in MCPs is likely critical for DifA function because shortening of this bundle can lead to constitutively active mutations.
PMCID: PMC3021235  PMID: 21131490
7.  Independence and interdependence of Dif and Frz chemosensory pathways in Myxococcus xanthus chemotaxis 
Molecular microbiology  2008;69(3):714-723.
Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation, respectively. DifA and FrzCD, the homologs of methyl-accepting chemoreceptors in the two pathways, were examined for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates that DifA may not undergo methylation but signals transmitting through DifA do modulate FrzCD methylation. Results also revealed that M. xanthus possesses Dif-dependent and Dif-independent PE sensing mechanisms. Previous studies showed that FrzCD methylation is decreased by negative chemostimuli but increased by attractants such as PE. Results here demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Dif-independent sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Dif-dependent PE signaling suppresses or diminishes the increase in FrzCD methylation to decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too quickly relative to the slow speed of M. xanthus movement.
PMCID: PMC2553899  PMID: 18554324
Myxococcus xanthus; DifA and FrzCD methylation; chemotaxis; phosphatidylethanolamine (PE); exopolysaccharide (EPS)
8.  The Hsp70-like StkA functions between T4P and Dif signaling proteins as a negative regulator of exopolysaccharide in Myxococcus xanthus 
PeerJ  2015;3:e747.
Myxococcus xanthus displays a form of surface motility known as social (S) gliding. It is mediated by the type IV pilus (T4P) and requires the exopolysaccharide (EPS) to function. It is clear that T4P retraction powers S motility. EPS on a neighboring cell or deposited on a gliding surface is proposed to anchor the distal end of a pilus and trigger T4P retraction at its proximal end. Inversely, T4P has been shown to regulate EPS production upstream of the Dif signaling pathway. Here we describe the isolation of two Tn insertions at the stk locus which had been known to play roles in cellular cohesion and formation of cell groups. An insertion in stkA (MXAN_3474) was identified based on its ability to restore EPS to a pilA deletion mutant. The stkA encodes a DnaK or Hsp70 homolog and it is upstream of stkB (MXAN_3475) and stkC (MXAN_3476). A stkB insertion was identified in a separate genetic screen because it eliminated EPS production of an EPS+ parental strain. Our results with in-frame deletions of these three stk genes indicated that the stkA mutant produced increased level of EPS while stkB and stkC mutants produced less EPS relative to the wild type. S motility and developmental aggregation were affected by deletions of stkA and stkB but only minimally by the deletion of stkC. Genetic epistasis indicated that StkA functions downstream of T4P but upstream of the Dif proteins as a negative regulator of EPS production in M. xanthus.
PMCID: PMC4319316  PMID: 25674362
Myxococcus xanthus; Type IV pilus (T4P); Dif pathway; Exopolysaccharide (EPS); Social motility; StkA/DnaK/Hsp70
9.  Myxococcus xanthus dif Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility 
Journal of Bacteriology  2000;182(20):5793-5798.
Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.
PMCID: PMC94702  PMID: 11004179
10.  CrdS and CrdA Comprise a Two-Component System That Is Cooperatively Regulated by the Che3 Chemosensory System in Myxococcus xanthus 
mBio  2011;2(4):e00110-11.
Myxococcus xanthus serves as a model organism for development and complex signal transduction. Regulation of developmental aggregation and sporulation is controlled, in part, by the Che3 chemosensory system. The Che3 pathway consists of homologs to two methyl-accepting chemotaxis proteins (MCPs), CheA, CheW, CheB, and CheR but not CheY. Instead, the output for Che3 is the NtrC homolog CrdA, which functions to regulate developmental gene expression. In this paper we have identified an additional kinase, CrdS, which directly regulates the phosphorylation state of CrdA. Both epistasis and in vitro phosphotransfer assays indicate that CrdS functions as part of the Che3 pathway and, in addition to CheA3, serves to regulate CrdA phosphorylation in M. xanthus. We provide kinetic data for CrdS autophosphorylation and demonstrate specificity for phosphotransfer from CrdS to CrdA. We further demonstrate that CheA3 destabilizes phosphorylated CrdA (CrdA~P), indicating that CheA3 likely acts as a phosphatase. Both CrdS and CheA3 control developmental progression by regulating the phosphorylation state of CrdA~P in the cell. These results support a model in which a classical two-component system and a chemosensory system act synergistically to control the activity of the response regulator CrdA.
While phosphorylation-mediated signal transduction is well understood in prototypical chemotaxis and two-component systems (TCS), chemosensory regulation of alternative cellular functions (ACF) has not been clearly defined. The Che3 system in Myxococcus xanthus is a member of the ACF class of chemosensory systems and regulates development via the transcription factor CrdA (chemosensory regulator of development) (K. Wuichet and I. B. Zhulin, Sci. Signal. 3:ra50, 2010; J. R. Kirby and D. R. Zusman, Proc. Natl. Acad. Sci. U. S. A. 100:2008–2013, 2003). We have identified and characterized a homolog of NtrB, designated CrdS, capable of specifically phosphorylating the NtrC homolog CrdA in M. xanthus. Additionally, we demonstrate that the CrdSA two-component system is negatively regulated by CheA3, the central processor within the Che3 system of M. xanthus. To our knowledge, this study provides the first example of an ACF chemosensory system regulating a prototypical two-component system and extends our understanding of complex regulation of developmental signaling pathways.
PMCID: PMC3147164  PMID: 21810965
11.  Divergent Regulatory Pathways Control A and S Motility in Myxococcus xanthus through FrzE, a CheA-CheY Fusion Protein†  
Journal of Bacteriology  2005;187(5):1716-1723.
Myxococcus xanthus moves on solid surfaces by using two gliding motility systems, A motility for individual-cell movement and S motility for coordinated group movements. The frz genes encode chemotaxis homologues that control the cellular reversal frequency of both motility systems. One of the components of the core Frz signal transduction pathway, FrzE, is homologous to both CheA and CheY from the enteric bacteria and is therefore a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY domain (FrzECheY). FrzECheY retains all of the highly conserved residues of the CheY superfamily of response regulators, including Asp709, analogous to phosphoaccepting Asp57 of Escherichia coli CheY. While in-frame deletion of the entire frzE gene caused both motility systems to show a hyporeversal phenotype, in-frame deletion of the FrzECheY domain resulted in divergent phenotypes for the two motility systems: hyperreversals of the A-motility system and hyporeversals of the S-motility system. To further investigate the role of FrzECheY in A and S motility, point mutations were constructed such that the putative phosphoaccepting residue, Asp709, was changed from D to A (and was therefore never subject to phosphorylation) or E (possibly mimicking constitutive phosphorylation). The D709A mutant showed hyperreversals for both motilities, while the D709E mutant showed hyperreversals for A motility and hyporeversal for S motility. These results show that the FrzECheY domain plays a critical signaling role in coordinating A and S motility. On the basis of the phenotypic analyses of the frzE mutants generated in this study, a model is proposed for the divergent signal transduction through FrzE in controlling and coordinating A and S motility in M. xanthus.
PMCID: PMC1064013  PMID: 15716443
12.  XerCD-Mediated Site-Specific Recombination Leads to Loss of the 57-Kilobase Gonococcal Genetic Island▿  
Journal of Bacteriology  2010;193(2):377-388.
Most strains of Neisseria gonorrhoeae carry the 57-kb gonococcal genetic island (GGI), as do a few strains of Neisseria meningitidis. The GGI is inserted into the chromosome at the dif site (difA) and is flanked by a partial repeat of the dif site (difB). Since dif is a sequence recognized by the site-specific recombinases XerC and XerD and the GGI shows evidence of horizontal acquisition, we hypothesized that the GGI may be acquired or lost by XerCD-mediated site-specific recombination. We show that while the GGI flanked by wild-type dif sites, difA and difB, is not readily lost from the gonococcal chromosome, the substitution of difB with another copy of difA allows the frequent excision and loss of the GGI. In mutants carrying two difA sites (difA+ difA+), the GGI can be detected as an extrachromosomal circle that exists transiently. A mutation of xerD diminished GGI excision from the chromosome of a difA+ difA+ strain, while mutations in recA or type IV secretion genes had no effect on the loss of the GGI. These data indicate that the GGI is maintained by the replication of the chromosome and that GGI excision and loss are dependent upon the dif sequence and xerD. The detection of a circular form of the GGI in a wild-type strain suggests that GGI excision may occur naturally and could function to facilitate GGI transfer. These data suggest a model of GGI excision and loss explaining the absence of the GGI from some gonococcal strains and the maintenance of variant GGIs in some gonococcal and meningococcal isolates.
PMCID: PMC3019826  PMID: 21075927
13.  Function of a Chemotaxis-Like Signal Transduction Pathway in Modulating Motility, Cell Clumping, and Cell Length in the Alphaproteobacterium Azospirillum brasilense▿ †  
Journal of Bacteriology  2008;190(19):6365-6375.
A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [Δ(cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.
PMCID: PMC2565992  PMID: 18641130
14.  The 3.2 Å resolution structure of a Receptor:CheA:CheW signaling complex defines overlapping binding sites and key residue interactions within bacterial chemosensory arrays 
Biochemistry  2013;52(22):3852-3865.
Bacterial chemosensory arrays are composed of extended networks of chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), the histidine kinase CheA, and the adaptor protein CheW. Models of these arrays have been developed from cryoelectron microscopy, crystal structures of binary and ternary complexes, NMR spectroscopy, mutational data and biochemical studies. A new 3.2 Å resolution crystal structure of a T. maritima MCP protein interaction region in complex with the CheA kinase-regulatory module (P4–P5) and adaptor protein CheW provides sufficient detail to define residue contacts at the interfaces formed among the three proteins. As in a previous 4.5 Å resolution structure, CheA-P5 and CheW interact through conserved hydrophobic surfaces at the ends of their β-barrels to from pseudo six-fold symmetric rings in which the two proteins alternate around the circumference. The interface between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous studies, whereas the related interface between CheW subdomain 1 and P5 subdomain 2 has only been observed in these ring assemblies. The receptor forms an unexpected structure in that the helical hairpin tip of each subunit has “unzipped” into a continuous α-helix; four such helices associate into a bundle and the tetramers bridge adjacent P5-CheW rings in the lattice through interactions with both P5 and CheW. P5 and CheW each bind a receptor helix with a groove of conserved hydrophobic residues between subdomains 1 and 2. P5 binds the receptor helix N-terminal to the tip region (lower site), whereas CheW binds the same helix with inverted polarity near the bundle end (upper site). Sequence comparisons among different evolutionary classes of chemotaxis proteins show that the binding partners undergo correlated changes at key residue positions that involve the lower site. Such evolutionary analyses argue that both CheW and P5 bind to the receptor tip at overlapping positions. Computational genomics further reveal that two distinct CheW proteins in Thermotogae utilize the analogous recognition motifs to couple different receptor classes to the same CheA kinase. Important residues for function previously identified by mutagenesis, chemical modification and biophysical approaches also map to these same interfaces. Thus, although the native CheW-receptor interaction is not observed in the present crystal structure, the bioinformatics and previous data predict key features of this interface. The companion study of the P5-receptor interface in native arrays (Piasta et al. (2013) Biochemistry; Companion paper) shows that, despite the non-native receptor fold in the present crystal structure, the local helix-in-groove contacts of the crystallographic P5-receptor interaction are present in native arrays and are essential for receptor regulation of kinase activity.
PMCID: PMC3694592  PMID: 23668907
Chemotaxis; histidine-kinase; x-ray crystallography; structure; ternary complex; signal transduction; transmembrane receptor; network; computational genomics
15.  Dual chemotaxis signaling pathways in Bacillus subtilis: a sigma D-dependent gene encodes a novel protein with both CheW and CheY homologous domains. 
Journal of Bacteriology  1994;176(9):2727-2735.
The alternative sigma factor, sigma D, activates the expression of genes required for chemotaxis and motility in Bacillus subtilis, including those encoding flagellin, hook-associated proteins, and the motor proteins. The sigma D protein is encoded in a large operon which also encodes the structural proteins for the basal body and homologs of the enteric CheW, CheY, CheA, and CheB chemotaxis proteins. We report the identification and molecular characterization of a novel chemotaxis gene, cheV. The predicted CheV gene product contains an amino-terminal CheW homologous domain linked to a response regulator domain of the CheY family, suggesting that either or both of these functions are duplicated. Transcription of cheV initiates from a sigma D-dependent promoter element both in vivo and in vitro, and expression of a cheV-lacZ fusion is completely dependent on sigD. Expression is repressed by nonpolar mutations in structural genes for the basal body, fliM or fliP, indicating that cheV belongs to class III in the B. subtilis flagellar hierarchy. The cheV locus is monocistronic and is located at 123 degrees on the B. subtilis genetic map near the previously defined cheX locus. A cheV mutant strain is motile but impaired in chemotaxis on swarm plates. Surprisingly, an insertion in the CheW homologous domain leads to a more severe defect than an insertion in the CheY homologous domain. The presence of dual pathways for chemotactic signal transduction is consistent with the residual signaling observed in previous studies of cheW mutants (D. W. Hanlon, L. Márques-Magaña, P. B. Carpenter, M. J. Chamberlin, and G. W. Ordal, J. Biol. Chem. 267:12055-12060, 1992).
PMCID: PMC205414  PMID: 8169223
16.  Adenylate Charge Regulates Sensor Kinase CheS3 To Control Cyst Formation in Rhodospirillum centenum 
mBio  2015;6(3):e00546-15.
Rhodospirillum centenum forms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che3 signaling cascade. The Che3 cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3 and CheR3, two CheW3 linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3 cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3 does not serve as a phosphor donor for CheY3; instead, CheA3 inhibits a CheS3→CheY3 two-component system by phosphorylating an inhibitory receiver domain of CheS3. In this study, we show that in addition to phosphorylation by CheA3, the phosphorylation state of CheS3 is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occur in vivo upon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicated in vitro, the phosphorylation level of CheS3 is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3 phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3.
Upon starvation, Rhodospirillum centenum undergoes a developmental process that forms metabolically dormant cysts, which withstand desiccation and nutritional limitation. This study explores the role of the cellular energy state as measured by the ratio of ATP to ADP as an important regulator of cyst formation in Rhodospirillum centenum. We show that R. centenum cells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When this in vivo level of energy starvation is simulated in vitro, CheS3 phosphorylation is reduced by 75%. This profound reduction in CheS3 autophosphorylation is contrasted with a much lower 25% decrease in CheA3 phosphorylation in response to a similar downward shift in ATP/(ATP + ADP). We argue that even though adenylate energy affects all ATP-dependent enzymes to an extent, the enhanced inhibition of CheS3 activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.
PMCID: PMC4436063  PMID: 25944862
17.  Regulation of expression of the pilA gene in Myxococcus xanthus. 
Journal of Bacteriology  1997;179(24):7748-7758.
Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.
PMCID: PMC179738  PMID: 9401034
18.  Identification of an Anchor Residue for CheA-CheY Interactions in the Chemotaxis System of Escherichia coli ▿  
Journal of Bacteriology  2011;193(15):3894-3903.
Transfer of a phosphoryl group from autophosphorylated CheA (P-CheA) to CheY is an important step in the bacterial chemotaxis signal transduction pathway. This reaction involves CheY (i) binding to the P2 domain of P-CheA and then (ii) acquiring the phosphoryl group from the P1 domain. Crystal structures indicated numerous side chain interactions at the CheY-P2 binding interface. To investigate the individual contributions of the P2 side chains involved in these contacts, we analyzed the effects of eight alanine substitution mutations on CheA-CheY binding interactions. An F214A substitution in P2 caused ∼1,000-fold reduction in CheA-CheY binding affinity, while Ala substitutions at other P2 positions had small effects (E171A, E178A, and I216A) or no detectable effects (H181A, D202A, D207A, and C213A) on binding affinity. These results are discussed in relation to previous in silico predictions of hot-spot and anchor positions at the CheA-CheY interface. We also investigated the consequences of these mutations for chemotaxis signal transduction in living cells. CheA(F214A) was defective in mediating localization of CheY-YFP to the large clusters of signaling proteins that form at the poles of Escherichia coli cells, while the other CheA variants did not differ from wild-type (wt) CheA (CheAwt) in this regard. In our set of mutants, only CheA(F214A) exhibited a markedly diminished ability to support chemotaxis in motility agar assays. Surprisingly, however, in FRET assays that monitored receptor-regulated production of phospho-CheY, CheA(F214A) (and each of the other Ala substitution mutants) performed just as well as CheAwt. Overall, our findings indicate that F214 serves as an anchor residue at the CheA-CheY interface and makes an important contribution to the binding energy in vitro and in vivo; however, loss of this contribution does not have a large negative effect on the overall ability of the signaling pathway to modulate P-CheY levels in response to chemoattractants.
PMCID: PMC3147499  PMID: 21642453
19.  Gliding Motility in Bacteria: Insights from Studies of Myxococcus xanthus 
Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.
PMCID: PMC103748  PMID: 10477310
20.  A CRISPR with Roles in Myxococcus xanthus Development and Exopolysaccharide Production 
Journal of Bacteriology  2014;196(23):4036-4043.
The Gram-negative soil bacterium Myxococcus xanthus utilizes its social (S) gliding motility to move on surfaces during its vegetative and developmental cycles. It is known that S motility requires the type IV pilus (T4P) and the exopolysaccharide (EPS) to function. The T4P is the S motility motor, and it powers cell movement by retraction. As the key regulator of the S motor, EPS is proposed to be the anchor and trigger for T4P retraction. The production of EPS is regulated in turn by the T4P in M. xanthus, and T4P− mutants are S− and EPS−. In this study, a ΔpilA strain (T4P− and EPS−) was mutagenized by a transposon and screened for EPS+ mutants. A pilA suppressor isolated as such harbored an insertion in the 3rd clustered regularly interspaced short palindromic repeat (CRISPR3) in M. xanthus. Evidence indicates that this transposon insertion, designated CRISPR3*, is a gain-of-function (GOF) mutation. Moreover, CRISPR3* eliminated developmental aggregation in both the wild-type and the pilA mutant backgrounds. Upstream of CRISPR3 are genes encoding the repeat-associated mysterious proteins (RAMPs). These RAMP genes are indispensable for CRISPR3* to affect development and EPS in M. xanthus. Analysis by reverse transcription (RT)-PCR suggested that CRISPR3* led to an increase in the processing of the RNA transcribed from CRISPR3. We propose that certain CRISPR3 transcripts, once expressed and processed, target genes critical for M. xanthus fruiting body development and EPS production in a RAMP-dependent manner.
PMCID: PMC4248871  PMID: 25201946
21.  Cluster II che Genes from Pseudomonas aeruginosa Are Required for an Optimal Chemotactic Response 
Journal of Bacteriology  2002;184(16):4374-4383.
Pseudomonas aeruginosa, a γ-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.
PMCID: PMC135244  PMID: 12142407
22.  Helicobacter pylori Possesses Two CheY Response Regulators and a Histidine Kinase Sensor, CheA, Which Are Essential for Chemotaxis and Colonization of the Gastric Mucosa 
Infection and Immunity  2000;68(4):2016-2023.
Infection of the mucous layer of the human stomach by Helicobacter pylori requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylori has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into cheY1, cheY2, and cheA in H. pylori strains N6 and SS1. Video tracking revealed that the wild-type H. pylori strain moves in short runs with frequent direction changes, in contrast to movement of cheY2, cheAY2, and cheAY2 cheY1 mutants, whose motion was more linear. The cheY1 mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1 nor cheAY2 mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1 failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of cheY1, cheY2, and cheA in motility and virulence of H. pylori.
PMCID: PMC97381  PMID: 10722597
23.  The Azospirillum brasilense Che1 Chemotaxis Pathway Controls Swimming Velocity, Which Affects Transient Cell-to-Cell Clumping 
Journal of Bacteriology  2012;194(13):3343-3355.
The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche.
PMCID: PMC3434747  PMID: 22522896
24.  Deciphering chemotaxis pathways using cross species comparisons 
Chemotaxis is the process by which motile bacteria sense their chemical environment and move towards more favourable conditions. Escherichia coli utilises a single sensory pathway, but little is known about signalling pathways in species with more complex systems.
To investigate whether chemotaxis pathways in other bacteria follow the E. coli paradigm, we analysed 206 species encoding at least 1 homologue of each of the 5 core chemotaxis proteins (CheA, CheB, CheR, CheW and CheY). 61 species encode more than one of all of these 5 proteins, suggesting they have multiple chemotaxis pathways. Operon information is not available for most bacteria, so we developed a novel statistical approach to cluster che genes into putative operons. Using operon-based models, we reconstructed putative chemotaxis pathways for all 206 species. We show that cheA-cheW and cheR-cheB have strong preferences to occur in the same operon as two-gene blocks, which may reflect a functional requirement for co-transcription. However, other che genes, most notably cheY, are more dispersed on the genome. Comparison of our operons with shuffled equivalents demonstrates that specific patterns of genomic location may be a determining factor for the observed in vivo chemotaxis pathways.
We then examined the chemotaxis pathways of Rhodobacter sphaeroides. Here, the PpfA protein is known to be critical for correct partitioning of proteins in the cytoplasmically-localised pathway. We found ppfA in che operons of many species, suggesting that partitioning of cytoplasmic Che protein clusters is common. We also examined the apparently non-typical chemotaxis components, CheA3, CheA4 and CheY6. We found that though variants of CheA proteins are rare, the CheY6 variant may be a common type of CheY, with a significantly disordered C-terminal region which may be functionally significant.
We find that many bacterial species potentially have multiple chemotaxis pathways, with grouping of che genes into operons likely to be a major factor in keeping signalling pathways distinct. Gene order is highly conserved with cheA-cheW and cheR-cheB blocks, perhaps reflecting functional linkage. CheY behaves differently to other Che proteins, both in its genomic location and its putative protein interactions, which should be considered when modelling chemotaxis pathways.
PMCID: PMC2829493  PMID: 20064255
25.  PilA localization affects extracellular polysaccharide production and fruiting body formation in Myxococcus xanthus 
Molecular microbiology  2010;76(6):1500-1513.
Myxococcus xanthus is a gram-negative bacterium capable of complex developmental processes involving vegetative swarming and fruiting body formation. Social (S-) gliding motility, one of the two motility systems employed by M. xanthus, requires at least two cell surface structures: type IV pili (TFP) and extracellular polysaccharides (EPS). Extended TFP which are composed of thousands of copies of PilA retract upon binding to EPS and thereby pull the cell forward. TFP also act as external sensor to regulate EPS production. In this study, we generated a random PilA mutant library and identified one derivative, SW1066, which completely failed to undergo developmental processes. Detailed characterization revealed that SW1066 produced very little EPS but wild-type amounts of PilA. These mutated PilA subunits, however, are unable to assemble into functional TFP despite their ability to localize to the membrane. By preventing the mutated PilA of SW1066 to translocate from the cytoplasm to the membrane, fruiting body formation and EPS production was restored to the levels observed in mutant strains lacking PilA. This apparent connection between PilA membrane accumulation and reduction in surface EPS implies that specific cellular PilA localization are required to maintain the EPS level necessary to sustain normal S-motilityin M. xanthus.
PMCID: PMC2935901  PMID: 20444090
Myxococcus xanthus; type four pili; PilA; extracellular polysaccharide

Results 1-25 (723145)